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Combinatorial Chemistry &
High Throughput Screening
ISSN: 1386-2073
Combinatorial Chemistry &
High Throughput Screening
Volume 9, Number 9, November 2006
Contents
A High-Throughput Mammalian Protein Expression,
Purification, Aliquoting and Storage Pipeline to Assemble
a Library of the Human Secretome Pp. 639-649
Thierry Battle, Bruno Antonsson, Georg Feger and Dominique
Besson
[Abstract]
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Synthesis and Screening of N-Alkyl Hydroxamates
for Inhibition of Cancer Cell Proliferation Pp. 651-661
Keith J. Stanger, Daniel Sliva, Jiahua Jiang and Viktor
Krchnák
[Abstract]
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Further Exploration of Antimicrobial Ketodihydronicotinic
Acid Derivatives by Multiple Parallel Syntheses Pp.
663-681
Jane B. Laursen, John Nielsen, Torsten Haack, Srinivas
Pusuluri, Sunil David, Rajalakshmi Balakrishna, Yibin Zeng,
Zhunkun Ma, Timothy B. Doyle and Lester A. Mitscher
[Abstract]
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Orphan Nuclear Receptors, Excellent Targets of
Drug Discovery Pp. 683-689
Yanhong Shi
[Abstract]
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Microwave-Assisted, Solid-Phase Synthesis of a
Chiral 1,2,3,4 Tetrahydroquinoline Library Pp. 691-701
Giordano Lesma, Bruno Danieli, Francesco Lodroni, Daniele
Passarella, Alessandro Sacchetti and Alessandra Silvani
[Abstract]
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Minimisation of Palladium Content in Suzuki Cross-Coupling
Reactions Pp. 703-710
Heather Tye, Mark Whittaker and Shailesh Ramdeehul
[Abstract]
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Article]
Parallel Analysis of v-Src Mutant Protein Function
Using Reverse Transfection Cell Arrays Pp. 711-718
Wanda Walczak, Nina H. Pipalia, Meenal Soni, A. Fawad
Faruqi, Harold Ralph, Frederick R. Maxfield and Brian L.Webb
[Abstract]
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Article]
Abstracts
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A High-Throughput Mammalian Protein Expression, Purification,
Aliquoting and Storage Pipeline to Assemble a Library of the
Human Secretome
Thierry Battle, Bruno Antonsson, Georg Feger and
Dominique Besson
In the post-human genome-sequencing era, the
availability of recombinant proteins has become crucial for
the identification of proteins with therapeutic potential.
Based upon bioinformatic coding predictions of the genes for
putative secreted proteins, we established a high-throughput
protein pipeline (HTPP) for the production of a subset of
the human secretome. The HTPP was based on a transient expression
system in HEK293-EBNA cells at 100 to 500 mL culture scale,
combined with an automated affinity purification procedure
targeting >75% purity. This was followed by a semi-automated
protein sample logistics to provide biologists with quality-controlled
and 96 well formatted protein aliquots amenable to cell-based
assays. Over a 4-year period, beginning in 2001, we performed
over 7,500 transfections representing over 2,200 registered
proteins, including both novel and reference proteins, at
an average production of 280 proteins/month with a peak production
of 320 proteins/month. All these proteins have been tested
in more than 50 different cell-based assays. This article
describes the major process steps and highlights the optimization
required to maintain novel protein production while supporting
both stock replenishment and scale-up productions.
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Synthesis and Screening of N-Alkyl
Hydroxamates for Inhibition of Cancer Cell Proliferation
Keith J. Stanger, Daniel Sliva, Jiahua Jiang and
Viktor Krchnák
A small library of N-alkylated amino acid-derived
sulfonamide hydroxamates was synthesized on solid phase and
tested for inhibition of proliferation of the highly invasive
breast cancer cell line MDA-MB-231. The most active compound
4317 inhibited cell growth at IC50
30 μM.
N-alkylation of N-H hydroxamate-based MMP
inhibitors, a modification known to eliminate MMP activity,
enhanced cell proliferation inhibition potency.
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Further Exploration of Antimicrobial Ketodihydronicotinic
Acid Derivatives by Multiple Parallel Syntheses
Jane B. Laursen, John Nielsen, Torsten Haack,
Srinivas Pusuluri, Sunil David, Rajalakshmi Balakrishna, Yibin
Zeng, Zhunkun Ma, Timothy B. Doyle and Lester A. Mitscher
A synthetic reexamination of a series of ketodihydronicotinic
acid class antibacterial agents was undertaken in an attempt
to improve their therapeutic potential. A convenient new synthesis
was developed involving hetero Diels-Alder chemistry producing
74 new analogs in a multiple parallel synthetic manner and
these were examined in vitro for their antimicrobial
potential. Several compounds demonstrated significant broad-spectrum
activity against clinically derived bacterial strains but
previously known 1-(2,4-difluorophenyl)-6-(4-dimethylaminophenyl)-4-pyridone-3-carboxylic
acid (7) remained the most potent compound in this class.
Cross-resistance with ciprofloxacin supported a commonality
of mode of action. Permiabilization of Escherichia coli
cells by polymyxin B significantly enhanced potency with these
agents suggesting that poor cellular uptake was primarily
responsible for the disappointing activity against bacteria
that some of the analogs exhibited
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Orphan Nuclear Receptors, Excellent Targets
of Drug Discovery
Yanhong Shi
To date, the pharmaceutical industry has placed
a considerable amount of interest in the discovery of drug
targets and diagnostics. One of the most challenging areas
of drug discovery today is the search for novel receptor-ligand
pairs. Nuclear receptors comprise a large superfamily of ligand-dependent
transcription factors that regulate the expression of genes
critical for a variety of biological processes, including
development, growth, differentiation, and homeostasis. Orphan
nuclear receptors, for which the ligands are not yet identified,
represent the most ancient component of the nuclear receptor
superfamily. Orphan nuclear receptors not only offer a unique
system to uncover novel signaling pathways that impact human
health, but also provide excellent targets of drug discoveries
for a variety of human diseases. This review highlights advances
made on ligand identification for orphan nuclear receptors
using transgenic mouse models, cell-based screening, direct
binding, structure-based assays, and computer-aided virtual
screening. With rapid advances in combinatorial chemistry
and high throughput screening, along with other modern technologies,
this field promises a bountiful harvest.
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Microwave-Assisted, Solid-Phase Synthesis
of a Chiral 1,2,3,4 Tetrahydroquinoline Library
Giordano Lesma, Bruno Danieli, Francesco Lodroni,
Daniele Passarella, Alessandro Sacchetti and Alessandra Silvani
The synergy of a solution-phase preparation of scaffolds
with a solid-phase combinatorial synthesis let to develop
an efficient route to a small library of chiral, highly functionalized
tetrahydroisoquinolines. Both loading on the Merrifield resin
and the key acid-catalyzed Pictet-Spengler condensation were
efficiently promoted by microwave irradiation. The library
was designed such that up to five points of diversity would
be potentially introduced, making the strategy in principle
suitable for generation of a large number of compounds.
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Minimisation of Palladium Content in Suzuki
Cross-Coupling Reactions
Heather Tye, Mark Whittaker and Shailesh Ramdeehul
A protocol for conducting Suzuki reactions in a
plate format amenable to use in library synthesis of biaryl
compounds has been developed. A key objective was to determine
reaction conditions which give biaryl products containing
<10 ppm of Pd for a wide variety of building blocks. A
Design of Experiments (DoE) approach for the identification
of an optimised set of reaction conditions was successfully
applied. The utility of the protocol developed has been demonstrated
by preparation of an array of 96 biaryl compounds in a parallel
fashion.
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Parallel Analysis of v-Src Mutant Protein
Function Using Reverse Transfection Cell Arrays
Wanda Walczak, Nina H. Pipalia, Meenal Soni, A.
Fawad Faruqi, Harold Ralph, Frederick R. Maxfield and Brian
L.Webb
The conversion of the genomic information produced
by the recent sequencing projects into a comprehensive understanding
of the human proteome has yet to occur. A new technology that
represents a potential bridge between genomics and proteomics
is reverse transfection. Reverse transfection cell microarrays
are produced by overlaying cDNA arrays with mammalian cells,
generating localized clusters of transfected cells with each
cluster overexpressing a unique protein. This miniaturized
cell-based microarray format affords parallel functional analysis
of thousands of cDNA constructs in a high throughput format.
In this report we document the development of a co-transfection
methodology for reverse transfection applications. The demonstrated
high co-transfection efficiency with a “marker”
plasmid encoding for GFP enables the identification of transfected
cells and eliminates the need for epitope-tagged constructs
in cell-based high throughput screening applications using
reverse transfection. This co-transfection method was used
to study in parallel the structure/function of multiple versions
of the v-Src protein using automated fluorescence microscopy.
The wild-type v-Src protein and four mutants having insertions
or deletions in the SH2 or SH3 domains displayed high levels
of tyrosine kinase activity in HEK293T cells. Three other
mutated v-Src proteins, including a kinase-dead version, were
shown to be defective for tyrosine kinase activity. This reverse
co-transfection approach is applicable for high throughput
screening of both cDNA libraries and positional scanning recombinant
protein libraries.
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